In the environment, the Gram-negative bacterium Legionella pneumophila colonizes biofilms and multiplies within various protozoa [1]. Upon transmission to the human lung, the bacteria replicate within alveolar macrophages, and may cause the severe pneumonia Legionnaires' disease [2, 3]. To establish its replicative niche, L. pneumophila prevents the fusion of its phagosome with lysosomes [4], and recruits early secretory vesicles at endoplasmic reticulum (ER) exit sites [5]. The resulting Legionella-containing vacuole (LCV) is characterized by the ER marker calnexin, the v-SNARE Sec22b, and the small GTPases Arf1 and Rab1 [6, 7]. Moreover, the LCV undergoes a transition from a tight to a spacious vacuole [8, 9] and eventually matures into an acidic vacuole [10], wherein the bacteria multiply independently of the bacterial Icm/Dot type IV secretion system (T4SS) [11]. The Icm/Dot T4SS, a conjugation machinery encoded by 25 different genes, is required for formation of the LCV [12, 13] as well as for modulation of phagocytosis [14, 15].
To date, more than 30 different Icm/Dot-secreted proteins have been identified as putative effectors, many of which form families of 2-6 paralogues [16-22]. The precise function of most of these proteins is not known, owing at least in part to the fact that L. pneumophila strains lacking even multiple family members do not show a phenotype with regard to intracellular replication [18, 20, 22]. However, the inability of Icm/dot mutants to direct phagocytosis and establish a LCV, suggests that at least some Icm/Dot-secreted proteins interfere with host cell phagocytosis or vesicle trafficking.
Indeed, the recently identified effectors LepA and LepB share homology with SNAREs and seem to promote the non-lytic release of vesicles containing L. pneumophila from amoebae [19]. The first Icm/Dot substrate to be functionally characterized, RalF, recruits the GTPase Arf1 to the LCV and acts as a guanine nucleotide exchange factor for the Arf family of small GTPases [16]. The Icm/Dot-secreted proteins SidC and its paralogue SdcA have no orthologues in the database, their function is unknown, and a sidC/sdcA double mutant shows no phenotype [18]. SdcA was recently identified in a screen using growth inhibition of yeast to select for putative L. pneumophila effector proteins [22]. To subvert host cell trafficking, the large number of Icm/Dot-secreted proteins is likely organized in a complex spatial and temporal manner.
In eukaryotic cells, the metabolism of phosphoinositide lipids is pivotal for the regulation of membrane dynamics during phagocytosis, endocytosis and exocytosis [23, 24]. Depending on phosphorylation at position 3, 4 and/or 5 of the D-myo-inositol ring, phosphoinositides recruit specific effectors to distinct membranes in a time- and organelle-dependent manner, thus coordinating intracellular membrane trafficking and actin remodelling, as well as receptor-mediated signal transduction. The central role of phosphoinositide second messengers is exploited by a number of intracellular bacterial pathogens [25], for example Shigella flexneri [26] and Salmonella enterica [27, 28] employ type III-secreted phosphoinositide phosphatases to modulate phosphoinositide metabolism during bacterial entry and intracellular replication.
Phosphoinositide metabolism is well characterized in the social amoeba Dictyostelium discoideum [29, 30], which supports Icm/Dot-dependent intracellular replication of L. pneumophila [31-33]. Here, we use a Dictyostelium strain lacking the class I PI(3) kinase (PI3K)-1 and -2 (ΔPI3K1/2; [34]) to demonstrate a role for phosphoinositide metabolism in phagocytosis, trafficking and intracellular replication of L. pneumophila. Furthermore, we identify Icm/Dot-secreted proteins, which specifically bind to PI(4)P, thus providing a mechanistic link between phosphoinositide metabolism and the subversion of host cell trafficking by L. pneumophila. 